Methods for detecting dehydroepiandrosterone by mass spectrometry

ABSTRACT

Provided are methods for determining the amount of underivatized dehydroepiandrosterone (DHEA) in a sample using mass spectrometry. The methods generally involve ionizing DHEA in a sample and detecting and quantifying the amount of the ion to determine the amount of DHEA in the sample.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

The application claims priority to U.S. application Ser. No. 12/207,428,filed Sep. 9, 2008, which is incorporated herein by reference in itsentirety, including all figures and tables.

FIELD OF THE INVENTION

The invention relates to the detection of dehydroepiandrosterone (DHEA).In a particular aspect, the invention relates to methods for detectingdehydroepiandrosterone by mass spectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Dehydroepiandrosterone (DHEA) [(3β-hydroxy-5-androsten-17-one)] is aweak androgen synthesized by the adrenal cortex. It has a short plasmahalf-life and is usually converted to dehydroepiandrosterone sulfate(DHEA-S). Excessive DHEA secretion can produce acne, hirsutism andvirilization via conversion to testosterone.

Mass spectrometric methods for measuring DHEA in a sample have beenreported. See, e.g., Chatman, K., et al., Anal Chem 1999, 71:2358-63;Liu, S., et al., Anal Chem 2003, 75:5835-46; Higashi, T., et al., JChromatogr B 2007, 195-201; Hsing, A., et al., Cancer EpidemiolBiomarkers Prev 2007, 16:1004-8; Labrie, F., et al., J Steroid BiochemMol Biol 2007, 107:57-69; Guo, T., et al., Arch Pathol Lab Med 2004,128:469-75; Guo, T., et al., Clinica Chemica Acta 2006, 372:76-82; andZou, P., et al., Food Addit Contain 2007, 24:1326-33. Guo, T., et al.and Zou, P., et al. recently reported liquid chromatography-tandem massspectrometry techniques of measuring DHEA in a sample by observation ofmass transitions from a precursor ion with a mass to charge ratio of 271to a fragment ion with a mass to charge ratio of 213, and a precursorion with a mass to charge ratio of 289.2 to a fragment ion with a massto charge ratio of 270.9, respectively.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount ofdehydroepiandrosterone (DHEA) in a sample by mass spectrometry,including tandem mass spectrometry. Preferably, the methods of theinvention do not include derivatizing DHEA in a sample prior to the massspectrometry analysis.

In one aspect, methods are provided for determining the amount ofunderivatized dehydroepiandrosterone (DHEA) in a test sample. Methods ofthis aspect include: (a) ionizing DHEA from a test sample to produce oneor more DHEA ions detectable by mass spectrometry, wherein the producedions comprise one or more ions selected from the group consisting ofions with a mass/charge ratio of 253.10±0.50, 197.13±0.50, and167.10±0.50; and (b) detecting the amount of one or more DHEA ions bymass spectrometry. Once the amount of the one or more DHEA ions ismeasured, the amount of DHEA ion(s) is related to the amount of DHEA inthe test sample. In some embodiments, the mass spectrometry is tandemmass spectrometry. In some embodiments, the methods further comprisepurifying DHEA in a test sample prior to mass spectrometry. In relatedembodiments, purifying comprises purifying DHEA in a test sample byliquid chromatography; preferably high performance liquid chromatography(HPLC). In some embodiments, purifying DHEA in a test sample comprisespurifying by high turbulence liquid chromatography (HTLC), followed byhigh performance liquid chromatography (HPLC); preferably with on-lineprocessing. In some embodiments, the test sample is body fluid;preferably plasma or serum. In some embodiments, the step of ionizingDHEA includes generating a precursor ion with a mass/charge ratio of253.10±0.50 and generating one or more fragment ions selected from thegroup consisting of ions with a mass/charge ratio of 197.13±0.50 and167.10±0.50. In some embodiments, the methods have a limit ofquantitation greater than or equal to 10 ng/dL but less than or equal to2000 ng/dL. In some embodiments, the amount of one or more DHEA ionsdetected by mass spectrometry is related to the presence or amount ofDHEA in a test sample by comparison to an internal standard; preferably2, 2, 4, 6, 6-d₅ testosterone. The features of the embodiments listedabove may be combined without limitation for use in methods of thepresent invention.

In a second aspect, methods are provided for determining the amount ofunderivatized dehydroepiandrosterone (DHEA) in a test sample by massspectrometry. Methods of this aspect include: (a) purifying DHEA in atest sample with high turbulence liquid chromatography (HTLC); (b)ionizing DHEA from the test sample to produce one or more DHEA ionsdetectable by mass spectrometry; and (c) detecting the amount of one ormore DHEA ions by mass spectrometry. In these methods, the amount of theDHEA ion(s) detected by mass spectrometry is related to the amount ofDHEA in the test sample. In some embodiments, the mass spectrometry istandem mass spectrometry. In some embodiments, purifying DHEA in a testsample comprises purifying with high performance liquid chromatography(HPLC). In related embodiments, HTLC and HPLC may be configured foron-line processing. In some embodiments, the test sample is a body fluidsample; preferably plasma or serum. In some embodiments, the DHEA ionsdetectable by mass spectrometry include one or more ions selected fromthe group consisting of ions with a mass/charge ratio of 253.10±0.50,197.13±0.50, and 167.10±0.50. In some embodiments, the step of ionizingDHEA includes generating a precursor ion with a mass/charge ratio of253.10±0.50 and generating one or more fragment ions selected from thegroup consisting of ions with a mass/charge ratio of 197.13±0.50 and167.10±0.50. In some embodiments, the methods have a limit ofquantitation greater than or equal to 10 ng/dL but less than or equal to2000 ng/dL. In some embodiments, the amount of one or more DHEA ionsdetected by mass spectrometry is related to the presence or amount ofDHEA in a test sample by comparison to an internal standard; preferably2, 2, 4, 6, 6-d₅ testosterone. The features of the embodiments listedabove may be combined without limitation for use in methods of thepresent invention.

In at third aspect, methods are provided for determining the amount ofunderivatized dehydroepiandrosterone (DHEA) in a test sample by tandemmass spectrometry. Methods of this aspect include: (a) purifying DHEAfrom the test sample by high turbulence liquid chromatography (HTLC);(b) generating a precursor ion of said DHEA having a mass/charge ratioof 253.10±0.50; (c) generating one or more fragment ions of saidprecursor ion, wherein at least one of said one or more fragment ionscomprise a fragment ion selected from the group of fragment ions havinga mass/charge ratio of 197.13±0.50 and 167.10±0.50; and (d) detectingthe amount of one or more of said ions generated in step (b) or (c) orboth and relating the detected ions to the amount of DHEA in the testsample. In some embodiments, purifying DHEA from a test sample furthercomprises high performance liquid chromatography (HPLC). In some relatedembodiments, HTLC and HPLC are configured for on-line processing. Insome embodiments, the test sample is body fluid sample; preferablyplasma or serum. In some embodiments, the methods have a limit ofquantitation greater than or equal to 10 ng/dL but less than or equal to2000 ng/dL. In some embodiments, the amount of one or more DHEA ionsdetected by mass spectrometry is related to the presence or amount ofDHEA in a test sample by comparison to an internal standard; preferably2, 2, 4, 6, 6-d₅ testosterone. The features of the embodiments listedabove may be combined without limitation for use in methods of thepresent invention.

Methods of the present invention involve the combination of liquidchromatography with mass spectrometry. In preferred embodiments, theliquid chromatography is HPLC. One preferred embodiment utilizes HPLCalone or in combination with one or more purification methods such asfor example HTLC and/or protein precipitation and filtration, to purifyDHEA in samples. In another preferred embodiment, the mass spectrometryis tandem mass spectrometry (MS/MS).

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in positive ion mode. Alternatively, massspectrometry is performed in negative ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI) or electrospray ionization (ESI), may be used in embodiments ofthe present invention. In certain preferred embodiments, DHEA ismeasured using APCI in positive ion mode.

In preferred embodiments, DHEA ions detectable in a mass spectrometerare selected from the group consisting of positive ions with amass/charge ratio (m/z) of 253.10±0.50, 197.10±0.50, and 167.10±0.50. Inparticularly preferred embodiments, a DHEA precursor ion has m/z of253.10±0.50, and one or more fragment ions are selected from the groupconsisting of ions having m/z of 197.10±0.50 and 167.10±0.50.

In preferred embodiments, a separately detectable internal standard isprovided in the sample, the amount of which is also determined in thesample. In these embodiments, all or a portion of both the endogenousDHEA and the internal standard present in the sample is ionized toproduce a plurality of ions detectable in a mass spectrometer, and oneor more ions produced from each are detected by mass spectrometry.

A preferred internal standard is 2, 2, 4, 6, 6-d₅ testosterone. Inpreferred embodiments, the internal standard ions detectable in a massspectrometer are selected from the group consisting of positive ionswith m/z of 294.10±0.50 and 100.06±0.50. In particularly preferredembodiments, an internal standard precursor ion has m/z of 294.10±0.50;and an internal standard fragment ion has m/z of 100.06±0.50.

In preferred embodiments, the presence or amount of the DHEA ion isrelated to the presence or amount of DHEA in a test sample by comparisonto a reference such as 2, 2, 4, 6, 6-d₅ testosterone.

In certain preferred embodiments, the limit of quantitation (LOQ) ofDHEA is within the range of 10 ng/dL to 2000 ng/dL, inclusive;preferably within the range of 10 ng/dL to 1000 ng/dL, inclusive;preferably within the range of 10 ng/dL to 500 ng/dL, inclusive;preferably within the range of 10 ng/dL to 250 ng/dL, inclusive;preferably within the range of 10 ng/dL to 100 ng/dL, inclusive;preferably within the range of 10 ng/dL to 50 ng/dL, inclusive;preferably within the range of 10 ng/dL to 20 ng/dL, inclusive;preferably about 10 ng/dL.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, “derivatizing” means reacting two molecules to form anew molecule. As used here, the names of derivatized forms of DHEAinclude an indication as to the nature of derivatization. For example,the sulfate derivative of DHEA is indicated by dehydroepiandrosteronesulfate (DHEA-S) and the oxime derivative of DHEA is indicated bydehydroepiandrosterone oxime (DHEA-O). Dehydroepiandrosterone (DHEA),without indication of derivatization, means underivatized DHEA.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedDHEA parent or daughter ions by mass spectrometry. Relative reduction asthis term is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “test sample” refers to any sample that maycontain DHEA. A preferred test sample may be a body fluid or tissue. Asused herein, the term “body fluid” means any fluid that can be isolatedfrom the body of an individual. For example, “body fluid” may includeblood, plasma, serum, bile, saliva, urine, tears, perspiration, and thelike.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, through capillary passageways, or through a single contiguouscolumn of solid support such as monolithic column. The retardationresults from the distribution of the components of the mixture betweenone or more stationary phases and the bulk fluid, (i.e., mobile phase),as this fluid moves relative to the stationary phase(s). Examples of“liquid chromatography” include reverse phase liquid chromatography(RPLC), high performance liquid chromatography (HPLC), and highturbulence liquid chromatography (HTLC).

As used herein, the term “high performance liquid chromatography” or“HPLC” refers to liquid chromatography in which the degree of separationis increased by forcing the mobile phase under pressure through astationary phase, typically a densely packed column.

As used herein, the term “high turbulence liquid chromatography” or“HTLC” refers to a form of chromatography that utilizes turbulent flowof the material being assayed through the column packing as the basisfor performing the separation. HTLC has been applied in the preparationof samples containing two unnamed drugs prior to analysis by massspectrometry. See, e.g., Zimmer et al., J Chromatogr A 854: 23-35(1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and5,772,874, which further explain HTLC. Persons of ordinary skill in theart understand “turbulent flow”. When fluid flows slowly and smoothly,the flow is called “laminar flow”. For example, fluid moving through anHPLC column at low flow rates is laminar. In laminar flow the motion ofthe particles of fluid is orderly with particles moving generally instraight lines. At faster velocities, the inertia of the water overcomesfluid frictional forces and turbulent flow results. Fluid not in contactwith the irregular boundary “outruns” that which is slowed by frictionor deflected by an uneven surface. When a fluid is flowing turbulently,it flows in eddies and whirls (or vortices), with more “drag” than whenthe flow is laminar. Many references are available for assisting indetermining when fluid flow is laminar or turbulent (e.g., TurbulentFlow Analysis: Measurement and Prediction, P. S. Bernard & J. M.Wallace, John Wiley & Sons, Inc., (2000); An Introduction to TurbulentFlow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm. As used in this context, the term“about” means±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%. In apreferred embodiment the analytical column contains particles of about 4μm in diameter.

As used herein, the term “on-line” or “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. Nos. 6,204,500,entitled “Mass Spectrometry From Surfaces;” 6,107,623, entitled “Methodsand Apparatus for Tandem Mass Spectrometry;” 6,268,144, entitled “DNADiagnostics Based On Mass Spectrometry;” 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis21:1164-67 (2000).

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser desorption thermal desorption is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatscauses the sample to transfer into the gas phase. The gas phase sampleis then drawn into the mass spectrometer.

As used herein, the term “limit of quantification”, “limit ofquantitation” or “LOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of 20% and an accuracy of 80% to 120%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as two times the RSD ofthe mean at the zero concentration.

As used herein, an “amount” of DHEA in a body fluid sample refersgenerally to an absolute value reflecting the mass of DHEA detectable involume of body fluid. However, an amount also contemplates a relativeamount in comparison to another DHEA amount. For example, an amount ofDHEA in a body fluid can be an amount which is greater than a control ornormal level of DHEA normally present.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows exemplary mass chromatograms of DHEA and 2, 2, 4,6, 6-d₅ testosterone (internal standard), respectively. Details arediscussed in Example 3.

FIG. 2 shows a plot of the coefficient of variation of assays of fivestandards each assayed five times to determine the limit of quantitationof the DHEA assay. Details are discussed in Example 5.

FIG. 3 shows the linearity of the quantitation of DHEA in seriallydiluted stock samples using an LC-MS/MS assay. Details are described inExample 6.

FIGS. 4A-C show the comparison of DHEA determined in serum samples andsamples from serum separator tubes (SST), EDTA tubes, and sodium heparintubes. Details are described in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

Methods of the present invention are described for measuring the amountof DHEA in a sample. More specifically, mass spectrometric methods aredescribed for detecting and quantifying DHEA in a test sample. Themethods may utilize liquid chromatography (LC), most preferably HPLC, toperform a purification of selected analytes, and combine thispurification with unique methods of mass spectrometry (MS), therebyproviding a high-throughput assay system for detecting and quantifyingDHEA in a test sample. The preferred embodiments are particularly wellsuited for application in large clinical laboratories for automated DHEAassay.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Particularly preferred samples includebodily fluids such as blood, plasma, serum, saliva, cerebrospinal fluid,or a tissue sample. Such samples may be obtained, for example, from apatient; that is, a living person, male or female, presenting oneself ina clinical setting for diagnosis, prognosis, or treatment of a diseaseor condition. The test sample is preferably obtained from a patient, forexample, blood serum or plasma. A sample volume of about 0.5 mL ispreferred; however, samples of about 0.1 mL can be analyzed.

The present invention contemplates kits for a DHEA quantitation assay. Akit for an DHEA quantitation assay of the present invention may includea kit comprising an internal standard, in amounts sufficient for atleast one assay. Typically, the kits will also include instructionsrecorded in a tangible form (e.g., contained on paper or an electronicmedium) for using the packaged reagents for use in a measurement assayfor determining the amount of DHEA.

Calibration and QC pools for use in embodiments of the present inventioncan be prepared using “stripped” plasma or serum (stripped of DHEA): forexample, double charcoal-stripped and delipidized serum. All sources ofhuman or non-human stripped plasma or serum should be checked to ensurethat they do not contain measurable amounts of DHEA.

Sample Preparation for Mass Spectrometry

Test samples may be stored below room temperature. Test samples(including controls) stored below room temperature are first allowed tocome to room temperature and mixed by mechanical vortex. Internalstandard may be added to the test samples at this point.

The samples may then be prepared for mass spectrometry by liquid-liquidor solid-phase extraction. Various methods may be used to enrich DHEArelative to other components in the sample (e.g. protein) prior massspectrometry, including for example, liquid chromatography, filtration,centrifugation, thin layer chromatography (TLC), electrophoresisincluding capillary electrophoresis, affinity separations includingimmunoaffinity separations, extraction methods including ethyl acetateor methanol extraction, and the use of chaotropic agents or anycombination of the above or the like.

Protein precipitation is one method of preparing a test sample,especially a biological test sample, such as serum or plasma. Suchprotein purification methods are well known in the art, for example,Polson et al., Journal of Chromatography B 2003, 785:263-275, describesprotein precipitation techniques suitable for use in methods of thepresent invention. Protein precipitation may be used to remove most ofthe protein from the sample leaving DHEA in the supernatant. The samplesmay be centrifuged to separate the liquid supernatant from theprecipitated proteins; alternatively the samples may be filtered toremove precipitated proteins. The resultant supernatant or filtrate maythen be applied directly to mass spectrometry analysis; or alternativelyto liquid chromatography and subsequent mass spectrometry analysis. Incertain embodiments, the use of protein precipitation such as forexample, formic acid protein precipitation, may obviate the need forhigh turbulence liquid chromatography (HTLC) or other on-line extractionprior to mass spectrometry or HPLC and mass spectrometry.

Accordingly, in some embodiments, protein precipitation, alone or incombination with one or more purification methods, may be used forpurification of DHEA prior to mass spectrometry. In these embodiments,the methods may involve (1) performing a protein precipitation of thesample of interest; and (2) loading the supernatant directly onto theLC-mass spectrometer without using on-line extraction or high turbulenceliquid chromatography (HTLC). Alternatively, the methods may involve (1)performing a protein precipitation of the sample of interest; and (2)loading the supernatant onto a HTLC using on-line extraction, forfurther purification prior to mass spectrometry.

One means of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including high-performance liquid chromatography (HPLC),rely on relatively slow, laminar flow technology. Traditional HPLCanalysis relies on column packing in which laminar flow of the samplethrough the column is the basis for separation of the analyte ofinterest from the sample. The skilled artisan will understand thatseparation in such columns is a diffusional process and may select HPLCinstruments and columns that are suitable for use with DHEA. Thechromatographic column typically includes a medium (i.e., a packingmaterial) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlesinclude a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the chemical moieties. One suitablebonded surface is a hydrophobic bonded surface such as an alkyl bondedsurface. Alkyl bonded surfaces may include C-4, C-8, C-12, or C-18bonded alkyl groups, preferably C-12 bonded groups. The chromatographiccolumn includes an inlet port for receiving a sample directly orindirectly from coupled SPE column and an outlet port for discharging aneffluent that includes the fractionated sample.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with a hydrophobic columnchromatographic system. In certain preferred embodiments, a C-12analytical column (e.g., a Synergi Max® C12 analytical column fromPhenomenex, Inc. (4 μm particle size, 75×3.0 mm or equivalent) is used.In certain preferred embodiments, HTLC and/or HPLC are performed usingHPLC Grade 0.1% aqueous formic acid and 100% methanol as the mobilephases.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, HTLC may be used for purification of DHEA prior tomass spectrometry. In such embodiments, samples may be extracted usingan HTLC extraction cartridge which captures the analyte, then eluted andchromatographed on a second HTLC column or onto an analytical HPLCcolumn prior to ionization. For example, samples extraction with an HTLCextraction cartridge may be accomplished with a large particle size (50μm) packed column. Sample eluted off of this column may then betransferred to an HPLC analytical column, such as a C-12 analyticalcolumn, for further purification prior to mass spectrometry. Because thesteps involved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

Detection and Quantitation by Mass Spectrometry

In various embodiments, DHEA present in a test sample may be ionized byany method known to the skilled artisan. Mass spectrometry is performedusing a mass spectrometer, which includes an ion source for ionizing thefractionated sample and creating charged molecules for further analysis.For example ionization of the sample may be performed by electronionization, chemical ionization, electrospray ionization (ESI), photonionization, atmospheric pressure chemical ionization (APCI),photoionization, atmospheric pressure photoionization (APPI), fast atombombardment (FAB), liquid secondary ionization (LSI), matrix assistedlaser desorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), inductively coupled plasma (ICP) and particle beamionization. The skilled artisan will understand that the choice ofionization method may be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

DHEA may be ionized in positive or negative mode. In preferredembodiments, DHEA is ionized by APCI in positive mode. In relatedpreferred embodiments, DHEA ion is in a gaseous state and the inertcollision gas is argon, nitrogen, or helium; preferably argon.

In mass spectrometry techniques generally, after the sample has beenionized, the positively charged or negatively charged ions therebycreated may be analyzed to determine a mass-to-charge ratio. Suitableanalyzers for determining mass-to-charge ratios include quadrupoleanalyzers, ion traps analyzers, and time-of-flight analyzers. The ionsmay be detected using several detection modes. For example, selectedions may be detected, i.e. using a selective ion monitoring mode (SIM),or alternatively, ions may be detected using a scanning mode, e.g.,multiple reaction monitoring (MRM) or selected reaction monitoring(SRM). Preferably, the mass-to-charge ratio is determined using aquadrupole analyzer. For example, in a “quadrupole” or “quadrupole iontrap” instrument, ions in an oscillating radio frequency fieldexperience a force proportional to the DC potential applied betweenelectrodes, the amplitude of the RF signal, and the mass/charge ratio.The voltage and amplitude may be selected so that only ions having aparticular mass/charge ratio travel the length of the quadrupole, whileall other ions are deflected. Thus, quadrupole instruments may act asboth a “mass filter” and as a “mass detector” for the ions injected intothe instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, molecular standards may be run withthe samples, and a standard curve constructed based on ions generatedfrom those standards. Using such a standard curve, the relativeabundance of a given ion may be converted into an absolute amount of theoriginal molecule. In certain preferred embodiments, an internalstandard is used to generate a standard curve for calculating thequantity of DHEA. Methods of generating and using such standard curvesare well known in the art and one of ordinary skill is capable ofselecting an appropriate internal standard. For example, an isotopicallylabeled steroid may be used as an internal standard; in certainpreferred embodiments the standard is 2, 2, 4, 6, 6-d₅ testosterone.Numerous other methods for relating the amount of an ion to the amountof the original molecule will be well known to those of ordinary skillin the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation is oftenused to generate the fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, DHEA is detected and/orquantified using MS/MS as follows. The samples are subjected to liquidchromatography, preferably HTLC (including solid phase extractionfollowed by HPLC); the flow of liquid solvent from the chromatographiccolumn enters the heated nebulizer interface of an MS/MS analyzer; andthe solvent/analyte mixture is converted to vapor in the heated tubingof the interface. The analyte (e.g., DHEA), contained in the nebulizedsolvent, is ionized by the corona discharge needle of the interface,which applies a large voltage to the nebulized solvent/analyte mixture.The ions, e.g. precursor ions, pass through the orifice of theinstrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 andQ3) are mass filters, allowing selection of ions (i.e., selection of“precursor” and “fragment” ions in Q1 and Q3, respectively) based ontheir mass to charge ratio (m/z). Quadrupole 2 (Q2) is the collisioncell, where ions are fragmented. The first quadrupole of the massspectrometer (Q1) selects for molecules with the mass to charge ratiosof DHEA. Precursor ions with the correct mass/charge ratios are allowedto pass into the collision chamber (Q2), while unwanted ions with anyother mass/charge ratio collide with the sides of the quadrupole and areeliminated. Precursor ions entering Q2 collide with neutral argon gasmolecules and fragment. This process is called collision activateddissociation (CAD). The fragment ions generated are passed intoquadrupole 3 (Q3), where the fragment ions of DHEA are selected whileother ions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of DHEA that may beused for selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte ofinterest. In certain embodiments, the area under the curves, oramplitude of the peaks, for fragment ion(s) and/or precursor ions aremeasured to determine the amount of DHEA. As described above, therelative abundance of a given ion may be converted into an absoluteamount of the original analyte, e.g., DHEA, using calibration standardcurves based on peaks of one or more ions of an internal molecularstandard, such as 2, 2, 4, 6, 6-d₅ testosterone.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1 Serum Sample and Reagent Preparation

Plasma samples were prepared by collecting blood in a Vacutainer tubewith no additives and allowed to clot for about 30 minutes at roomtemperature. Samples were then centrifuged and the serum separated fromthe cells. Samples that exhibited gross hemolysis were excluded.

Two DHEA stock solutions were prepared. A DHEA stock standard solutionof 1 mg/mL in methanol was prepared in a volumetric flask. A portion ofthe DHEA stock standard solution was then diluted 100× to prepare a DHEAintermediate stock standard solution of 1,000,000 ng/dL in methanol. Theintermediate stock standard solution was used to prepare two workingstandards: a DHEA working standard of 20,000 ng/dL in methanol, and aDHEA working standard of 10,000 ng/dL in stripped serum.

2, 2, 4, 6, 6-d₅ testosterone (CDN Isotopes, Cat. No. D-4000, orequivalent) was used to prepare a 1.0 mg/mL in deuterated methanol 2, 2,4, 6, 6-d₅ testosterone internal standard stock solution, which was usedto prepare a 10,000 ng/dL in deuterated methanol 2, 2, 4, 6, 6-d₅testosterone intermediate internal standard stock solution: 100 μl, ofthe 2, 2, 4, 6, 6-d₅ testosterone internal standard stock solution wasdiluted to volume with DI water in a 10 mL volumetric flask. The 2, 2,4, 6, 6-d₅ testosterone intermediate internal standard stock solutionwas used to prepare 10 ng/mL in water 2, 2, 4, 6, 6-d₅ testosteroneintermediate internal standard working solution: 200 μL 2, 2, 4, 6, 6-d₅testosterone intermediate internal standard stock solution was dilutedto volume with DI water in a 200 mL volumetric flask

Example 2 Extraction of DHEA from Samples Using Liquid Chromatography

Room temperature standards, controls, and patient samples were preparedfor liquid chromatography (LC) by first mixing by mechanical vortex.

200 μL it of each vortexed standard, control, and patient sample wasthen transferred to a well of a 96-well plate. 400 μL of 20% formic acidand 100 μL of 10 ng/mL, 2, 2, 4, 6, 6-d₅ testosterone internal standardwere then added to each. The plates were then vortexed and incubated atroom temperature for 30 minutes before being loaded into an autosamplerdrawer.

Sample injection was performed with a Cohesive Technologies Aria TLX-1HTLC system using Aria OS V1.5 or newer software. An autosampler washsolution was prepared using 60% acetonitrile, 30% isopropanol, and 10%acetone (v/v).

The HTLC system automatically injected 100 μL of the above preparedsamples into a TurboFlow column (50×1.0 mm, 50 μm C18 column fromCohesive Technologies) packed with large particles. The samples wereloaded at a high flow rate (5.0 mL/min, loading reagent 0.1% formicacid) to create turbulence inside the extraction column. This turbulenceensured optimized binding of DHEA to the large particles in the columnand the passage of residual protein and debris to waste.

Following loading, the flow direction was reversed and the sample elutedoff to the analytical column (Phenomenex, Inc. analytical column,Synergi Max® C12 column, 4 μm particle size, 75×3.0 mm). A binary HPLCgradient was applied to the analytical column, to separate DHEA fromother analytes contained in the sample. Mobile phase A was 0.1% formicacid and mobile phase B was 100% methanol. The HPLC gradient startedwith a 5% organic gradient which ramped to 95% in approximately 5.5minutes. The separated sample was then subjected to MS/MS forquantitation of DHEA.

The specificity of the DHEA against similar analytes was determined forthe following compounds (each at a concentration of 100 ng/dL instripped serum): testosterone, estrone, estradiol, estriol,pregnenolone, DHEA-S, androstenedione, and 17-OH pregnenolone. Nosignificant interference for any of these compounds was observed.

Example 3 Detection and Quantitation of DHEA by MS/MS

MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS system(Thermo Electron Corporation). The following software programs all fromThermoElectron were used in the Examples described herein: Quantum TuneMaster V1.2 or newer, Xcalibur V1.4 SK1 or newer, TSQ Quantum 1.4 ornewer, and LCQuan V 2.0 with SP1 or newer. Liquid solvent/analyteexiting the analytical column flowed to the heated nebulizer interfaceof a Thermo Finnigan MS/MS analyzer. The solvent/analyte mixture wasconverted to vapor in the heated tubing of the interface. Analytes inthe nebulized solvent were ionized by APCI.

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of 253.10±0.50 m/z. Ions entering Quadrupole 2 (Q2)collided with argon gas to generate ion fragments, which were passed toquadrupole 3 (Q3) for further selection. Simultaneously, the sameprocess using isotope dilution mass spectrometry was carried out with aninternal standard, 2, 2, 4, 6, 6-d₅ testosterone. The following masstransitions were used for detection and quantitation during validationon positive polarity.

TABLE 1 Mass Transitions for DHEA (Positive Polarity) Analyte PrecursorIon (m/z) Product Ion (m/z) DHEA 253.10 167.10 and 197.13 2,2,4,6,6-d₅testosterone 294.10 100.06 (internal standard)

Exemplary chromatograms for DHEA and 2, 2, 4, 6, 6-d₅ testosterone(internal standard) are found in FIG. 1.

Example 4 Intra-Assay and Inter-Assay Precision and Accuracy

Three quality control (QC) pools were prepared from analyte-strippedhuman serum (Golden West Biologicals, Temecula, Calif.) spiked with DHEAto a concentration of 30, 300, and 900 ng/dL, to cover the presumptivereportable range of the assay.

Ten aliquots from each of the three QC pools were analyzed in a singleassay to determine the coefficient of variation (CV (%)) of a samplewithin an assay. The following values were determined:

TABLE 2 Intra-Assay Variation and Accuracy Level I Level II Level III(30 ng/dL) (300 ng/dL) (900 ng/dL) Mean (ng/dL) 28.87 296.73 874.12Standard Deviation (ng/dL) 3.90 15.42 28.41 CV (%) 13.51% 5.19% 3.25%Accuracy (%) 96.23% 98.91% 97.12%

Ten aliquots from each of the three QC pools were assayed over ten daysto determine the coefficient of variation (CV (%)) between assays. Thefollowing values were determined:

TABLE 3 Inter-Assay Variation and Accuracy Level I Level II Level III(30 ng/dL) (300 ng/dL) (900 ng/dL) Mean (ng/dL) 29.98 295.32 926.92Standard Deviation (ng/dL) 4.27 22.83 66.17 CV (%) 14.26% 7.73% 7.13%Accuracy (%) 99.96% 98.44% 102.99%

Example 5 Analytical Sensitivity: Limit of Detection (LOD) and Limit ofQuantitation (LOQ)

The LOQ is the point where measurements become quantitativelymeaningful. The analyte response at this LOQ is identifiable, discreteand reproducible with a precision of 20% and an accuracy of 80% to 120%.The LOQ was determined by assaying analyte-stripped serum specimensspiked with DHEA concentrations of 5, 10, 20, 50, and 100 ng/dL (fivereplicates at each level) then determining the CV. The results wereplotted (as seen in FIG. 2) and the LOQ was determined from the curve tobe 10.0 ng/dL.

The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as two standarddeviations from the zero concentration. To determine the LOQ for theDHEA assay, blank samples of charcoal-stripped serum were run in tenreplicates. The results of these assays were statistically analyzed witha mean value of 1.43 ng/dL, a SD of 0.44, and a CV of 30.6%. Thus, theLOD for the DHEA was 2.30 ng/dL.

Example 6 Assay Reportable Range and Linearity

To establish the linearity of DHEA detection in the assay, threeseparate assays, each including one blank assigned as zero standard andeight spiked serum standards at concentrations ranging from a zerostandard to 2000 ng/dL were performed. The correlation value of theconcentration range tested (0 to 2000 ng/dL) was greater than 0.995. Agraph showing the linearity of the standard curve up to 2000 ng/dL isshown in FIG. 3.

Example 7 Matrix Specificity

Matrix specificity was evaluated by diluting patient serum samplesthree-fold and five-fold with the following matrices: analyte-strippedserum (charcoal stripped serum, Cat. No. SP1070, Golden WestBiologicals, Inc.), normal human defibrinated serum (Cat. No. 1101-00,Biocell Labs, Carson, Calif. 90746, or equivalent), and deionized (DI)water. Four serum samples were spiked with the following concentrationsof DHEA: 119.3 ng/dL, 72.4 ng/dL, 913 ng/dL, and 303 ng/dL. The spikedserums were then diluted 3× and 5× with the above matrices and analyzed.The study indicated that stripped serum is preferred for dilution ofsamples with analyte values above the linear range. The results of thisstudy are presented in Table 4.

TABLE 4 Matrix Specificity of DHEA Expected Stripped BiocellConcentration Serum Serum DI Water Dilutions (ng/dL) (ng/dL) (ng/dL)(ng/dL) Sample 1 — 119.3 3x 39.8 42.78 103.1 38.62 5x 23.86 19.13 55.5812.61 Sample 2 — 72.4 3x 24.1 24.10 65.66 18.45 5x 14.47 16.60 52.9810.52 Sample 3 — 913 3x 304.62 307.28 376.11 351.93 5x 182.77 163.77220.63 196.33 Sample 4 — 303 3x 101.15 84.89 132.9 118.63 5x 60.69 68.681.25 58.69

Example 8 Recovery

Pooled human serum or plasma from specimens older than one month wereused. The pool was split into three groups spiked with DHEA to aconcentration of 254, 470, and 1010 ng/dL.

A recovery study of these DHEA spiked samples was performed (inquadruplicate for each concentration). Absolute recovery was calculatedby dividing the DHEA concentration detected in the pooled samples by theexpected DHEA concentration in samples. The mean recoveries were 89.22%,92.19%, and 90.33%, respectively, giving an overall recovery of 90.58%.All recoveries were acceptable, i.e., within the range of 80% to 120%.

Example 9 Specimen Studies

Specimens derived from sample collection tubes with no additives (forserum), serum separator tubes (SST), EDTA tubes, or sodium heparin tubes(50 samples each, 25 from males and 25 from females) were tested for theapplicability of the instant methods to various sample types.

FIGS. 4A-C show comparisons of the DHEA determination for serum samplesfrom collection tubes with no additives and samples from the othercollection tubes listed above (SST, EDTA, and sodium heparin). WhileDHEA was detectable in all tested sample types, the DHEA levels detectedin samples from EDTA tubes were most similar to DHEA levels detected insamples from collection tubes with no additives (see FIG. 4B).

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A method for determining the amount of underivatizeddehydroepiandrosterone (DHEA) in a test sample, said method comprising:a. ionizing DHEA from said test sample by atmospheric pressure chemicalionization (APCI) to produce one or more DHEA ions detectable by massspectrometry, wherein one of said ions has a mass/charge ratio (m/z) of253.10±0.50; and b. detecting the amount of one or more DHEA ions bymass spectrometry, wherein the detected amount of DHEA ion(s) is relatedto the amount of underivatized DHEA in said test sample.
 2. The methodof claim 1, wherein said mass spectrometry is tandem mass spectrometry.3. The method of claim 2, wherein the ions detected in step (b) compriseone or more DHEA fragment ions of said 253.10±0.50 m/z DHEA ion.
 4. Themethod of claim 3, wherein said fragment ions are selected from thegroup of ions with m/z of 197.13±0.50 and 167.10±0.50.
 5. The method ofclaim 1, further comprising purifying underivatized DHEA in said testsample prior to ionizing DHEA.
 6. The method of claim 5, wherein saidpurifying comprises purifying with liquid chromatography.
 7. The methodof claim 6, wherein said liquid chromatography comprises highperformance liquid chromatography (HPLC).
 8. The method of claim 5,wherein said purifying comprises purifying with an extraction column. 9.The method of claim 8, wherein said extraction column is a highturbulence liquid chromatography (HTLC) column.
 10. The method of claim1, wherein said test sample is a body fluid.
 11. The method of claim 1,wherein said test sample is plasma or serum.
 12. The method of claim 1,wherein said method has a limit of quantitation within the range of 10ng/dL to 2000 ng/dL, inclusive.
 13. A method for determining the amountof underivatized dehydroepiandrosterone (DHEA) by mass spectrometry in aserum or plasma sample when taken from a human, said method comprising:a. ionizing DHEA from said test sample by atmospheric pressure chemicalionization (APCI) to produce one or more DHEA ions detectable by massspectrometry; and b. detecting the amount of one or more DHEA ions bymass spectrometry, wherein the detected amount of DHEA ion(s) is relatedto the amount of underivatized DHEA in said sample when taken from ahuman.
 14. The method of claim 13, wherein DHEA in said sample ispurified prior to ionization.
 15. The method of claim 14, whereinpurifying comprises purifying DHEA with an extraction column.
 16. Themethod of claim 15, wherein said extraction column is a high turbulenceliquid chromatography (HTLC) column.
 17. The method of claim 13, whereinthe one or more DHEA ions produced by the ionization of step a)comprises DHEA ions with a mass/charge ratio (m/z) of 253.10±0.50. 18.The method of claim 13, wherein said mass spectrometry is tandem massspectrometry.
 19. The method of claim 18, wherein the ionization of step(b) comprises producing a DHEA precursor ion with a mass/charge ratio(m/z) of 253.10±0.50 and producing one or more fragment ions of saidDHEA precursor ion.
 20. The method of claim 19, wherein said fragmentions are selected from the group consisting of ions with a mass/chargeratio (m/z) of 197.13±0.50 and 167.10±0.50.
 21. The method of claim 13,wherein said purifying further comprises purifying with high performanceliquid chromatography (HPLC).
 22. The method of claim 21, wherein saidHTLC and said HPLC are configured for on-line processing.
 23. The methodof claim 13, wherein said test sample is a body fluid sample.
 24. Themethod of claim 13, wherein said test sample is plasma or serum.
 25. Themethod of claim 13, wherein said method has a limit of quantitationwithin the range of 10 ng/dL to 2000 ng/dL, inclusive.
 26. A method fordetermining the amount of underivatized dehydroepiandrosterone (DHEA) ina test sample by tandem mass spectrometry said method comprising: a.purifying DHEA in said test sample by high turbulence liquidchromatography (HTLC); b. ionizing DHEA by atmospheric pressure chemicalionization (APCI) to produce a DHEA precursor ion with a mass/chargeratio of 253.10±0.50; c. producing one or more fragment ions of saidDHEA precursor ion; and d. detecting the amount of one or more of saidions produced in step (b) or (c) or both and relating the detected ionsto the amount of underivatized DHEA in said test sample.
 27. The methodof claim 26, wherein at least one of said one or more fragment ionscomprise a fragment ion selected from the group of fragment ions havinga mass/charge ratio of 197.13±0.50 and 167.10±0.50.
 28. The method ofclaim 26, wherein purifying DHEA in said test sample further compriseshigh performance liquid chromatography (HPLC).
 29. The method of claim26, wherein said test sample is a body fluid sample.
 30. The method ofclaim 26, wherein said test sample is plasma or serum.
 31. The method ofclaim 26, wherein said method has a limit of quantitation within therange of 10 ng/dL, to 2000 ng/dL, inclusive.